JP2010528041A5

JP2010528041A5 -

Info

Publication number: JP2010528041A5
Application number: JP2010509534A
Authority: JP
Filing date: 2008-05-21
Publication date: 2012-07-05
Anticipated expiration: 2028-05-21

Description

本発明は、ＲＮＡ誘導遺伝子調節または遺伝子分析、特に、ＲＮＡ干渉に対して使用するためのＲＮＡ鎖に取り込まれた１個または複数個のヒドロキシメチル置換モノマーを有するＲＮＡ複合体を提供する。そのため、本発明の目的は、通常使用されるＲＮＡ複合体と比較して、オフターゲット効果が低いＲＮＡ複合体を提供することである。別の目的は、インターフェロン応答性の低減を誘導するＲＮＡ複合体を提供することである。さらに別の目的は、細胞培養物中で、またはｉｎｖｉｖｏで酵素的分解への安定性に関する特性を改善したＲＮＡ複合体を提供することである。さらに別の目的は、未改変ＲＮＡ複合体に対して、細胞培養物中で、またはｉｎｖｉｖｏで、遺伝子調節機能、例えば、遺伝子サイレンシング効果の強化を示すＲＮＡ複合体を提供することである。さらなる目的は、特定の器官または組織に対して標的とされるＲＮＡ複合体、および細胞膜を通過することができるＲＮＡ複合体を提供することである。本発明は、ヒドロキシメチル置換モノマーのオリゴヌクレオチドへの取り込みに適切なモノマー、およびそれらの合成のための方法も提供する。
本発明は、例えば以下の項目を提供する。
（項目１）
非環式ヌクレオチドモノマーを含むオリゴヌクレオチド。
（項目２）
前記非環式ヌクレオチドモノマーは、２’−３’−セコ−ヌクレオチドモノマーである、項目２に記載のオリゴヌクレオチド。
（項目３）
前記非環式ヌクレオチドモノマーは、以下のモノマーＤ、Ｆ、Ｇ、Ｈ、Ｉ、およびＪから成る群より選択され、

式中、Ｒは、Ｈ、アルキル、コレステロール誘導体、フルオロフォア、ポリアミン、脂肪酸、アミノ酸、単糖類、およびポリペプチドから成る群より選択される、項目１〜２のいずれかに記載のオリゴヌクレオチド。
（項目４）
１５〜４０個のヌクレオチドモノマーを含む、項目１〜３のいずれかに記載のオリゴヌクレオチド。
（項目５）
１〜５個の非環式ヌクレオチドモノマーを含む、項目１〜５のいずれかに記載のオリゴヌクレオチド。
（項目６）
４個未満の連続的ＤＮＡモノマーを含む、項目１〜５のいずれかに記載のオリゴヌクレオチド。
（項目７）
５０％を上回るＲＮＡモノマーを含む、項目１〜６のいずれかに記載のオリゴヌクレオチド。
（項目８）
２’−Ｏ−アルキル−ＲＮＡモノマー、２’−アミノ−ＤＮＡモノマー、２’−フルオロ−ＤＮＡモノマー、ＬＮＡモノマー、ＰＮＡモノマー、ＨＮＡモノマー、ＡＮＡモノマー、ＦＡＮＡモノマー、ＣｅＮＡモノマー、ＥＮＡモノマー、ＤＮＡモノマー、およびＩＮＡモノマーから成る群より選択されるヌクレオチド類似体をさらに含む、項目１〜７のいずれかに記載のオリゴヌクレオチド。
（項目９）
少なくとも２つのＬＮＡヌクレオチド類似体を含む、項目１〜８のいずれかに記載のオリゴヌクレオチド。
（項目１０）
ホスホロチオエート結合またはボラノホスフェート結合を含む、項目１〜９のいずれかに記載のオリゴヌクレオチド。
（項目１１）
前記オリゴヌクレオチドは、該オリゴヌクレオチドに相補的な標的ｍＲＮＡのＲＩＳＣ依存性翻訳抑制または分解を媒介することが可能である、項目１〜１０のいずれかに記載のオリゴヌクレオチド。
（項目１２）
項目１〜１１のいずれかに記載の少なくとも１つのオリゴヌクレオチドを含む、ＲＮＡ二本鎖。
（項目１３）
１５〜４０個の多数の塩基対を含む、項目１２に記載のＲＮＡ二本鎖。
（項目１４）
１４〜２６個の多数の塩基対を含む、項目１２に記載のＲＮＡ二本鎖。
（項目１５）
少なくとも１つの突出を含む、項目１２〜１４のいずれかに記載のＲＮＡ二本鎖。
（項目１６）
前記突出の長さは、１〜１４個のヌクレオチドである、項目１５に記載のＲＮＡ二本鎖。
（項目１７）
少なくとも１つの３’突出を含む、項目１２〜１５のいずれかに記載のＲＮＡ二本鎖。
（項目１８）
前記少なくとも１つの３’突出の長さは、１〜１４個のヌクレオチドである、項目１７に記載のＲＮＡ二本鎖。
（項目１９）
少なくとも１つの平滑末端を含む、項目１２に記載のＲＮＡ二本鎖。
（項目２０）
前記ＲＮＡ二本鎖は、標的ｍＲＮＡに相補的なヌクレオチド配列を含むオリゴヌクレオチドを含み、ここで、該ＲＮＡ二本鎖は、該標的ｍＲＮＡの発現を低減させる、項目１２〜１９のいずれかに記載のＲＮＡ二本鎖。
（項目２１）
前記ＲＮＡ二本鎖は、非環式モノマーの代わりに天然ＲＮＡモノマーを有する同一のＲＮＡ二本鎖と比較して、オフターゲット効果を低下させる、項目１２〜２０のいずれかに記載のＲＮＡ二本鎖。
（項目２２）
前記ＲＮＡ二本鎖は、非環式モノマーの代わりに天然ＲＮＡモノマーを有する同一のＲＮＡ二本鎖と比較して、有効性を増大または持続させる、項目１２〜２１のいずれかに記載のＲＮＡ二本鎖。
（項目２３）
前記ＲＮＡ二本鎖は、非環式モノマーの代わりに天然ＲＮＡモノマーを有する同一のＲＮＡ二本鎖と比較して、安定性を改善させる、項目１２〜２２のいずれかに記載のＲＮＡ二本鎖。
（項目２４）
前記ＲＮＡ二本鎖は、非環式モノマーの代わりに天然ＲＮＡモノマーを有する同一のＲＮＡ二本鎖と比較して、免疫刺激を低減させる、項目１２〜２３のいずれかに記載のＲＮＡ二本鎖。
The present invention provides RNA complexes having one or more hydroxymethyl substituted monomers incorporated into the RNA strand for use against RNA-induced gene regulation or gene analysis, particularly RNA interference. Therefore, an object of the present invention is to provide an RNA complex having a low off-target effect as compared with a commonly used RNA complex. Another object is to provide RNA complexes that induce a reduction in interferon responsiveness. Yet another object is to provide RNA complexes with improved properties regarding stability to enzymatic degradation in cell culture or in vivo. Yet another object is to provide RNA complexes that exhibit enhanced gene regulatory functions, eg, gene silencing effects, in cell culture or in vivo relative to unmodified RNA complexes. A further object is to provide RNA complexes that are targeted to specific organs or tissues and that can cross cell membranes. The present invention also provides monomers suitable for incorporation of hydroxymethyl substituted monomers into oligonucleotides and methods for their synthesis.
For example, the present invention provides the following items.
(Item 1)
An oligonucleotide comprising an acyclic nucleotide monomer.
(Item 2)
The oligonucleotide according to item 2, wherein the acyclic nucleotide monomer is a 2′-3′-seco-nucleotide monomer.
(Item 3)
The acyclic nucleotide monomer is selected from the group consisting of the following monomers D, F, G, H, I, and J;

The oligonucleotide according to any one of items 1 to 2, wherein R is selected from the group consisting of H, alkyl, cholesterol derivative, fluorophore, polyamine, fatty acid, amino acid, monosaccharide, and polypeptide.
(Item 4)
4. The oligonucleotide according to any of items 1 to 3, comprising 15 to 40 nucleotide monomers.
(Item 5)
6. The oligonucleotide according to any of items 1 to 5, comprising 1 to 5 acyclic nucleotide monomers.
(Item 6)
6. The oligonucleotide according to any of items 1 to 5, comprising less than 4 continuous DNA monomers.
(Item 7)
7. The oligonucleotide according to any of items 1 to 6, comprising more than 50% RNA monomer.
(Item 8)
2′-O-alkyl-RNA monomer, 2′-amino-DNA monomer, 2′-fluoro-DNA monomer, LNA monomer, PNA monomer, HNA monomer, ANA monomer, FANA monomer, CeNA monomer, ENA monomer, DNA monomer, 8. The oligonucleotide according to any of items 1-7, further comprising a nucleotide analog selected from the group consisting of INA monomers.
(Item 9)
9. The oligonucleotide according to any of items 1-8, comprising at least two LNA nucleotide analogues.
(Item 10)
The oligonucleotide according to any of items 1 to 9, comprising a phosphorothioate bond or a boranophosphate bond.
(Item 11)
The oligonucleotide according to any one of items 1 to 10, wherein the oligonucleotide is capable of mediating RISC-dependent translational repression or degradation of a target mRNA complementary to the oligonucleotide.
(Item 12)
An RNA duplex comprising at least one oligonucleotide according to any of items 1-11.
(Item 13)
13. The RNA duplex of item 12, comprising a large number of 15-40 base pairs.
(Item 14)
Item 13. The RNA duplex of item 12, comprising a large number of 14 to 26 base pairs.
(Item 15)
15. The RNA duplex according to any of items 12-14, comprising at least one overhang.
(Item 16)
Item 16. The RNA duplex according to Item 15, wherein the overhang has a length of 1 to 14 nucleotides.
(Item 17)
16. The RNA duplex according to any of items 12-15, comprising at least one 3 'overhang.
(Item 18)
Item 18. The RNA duplex of item 17, wherein the length of the at least one 3 'overhang is 1 to 14 nucleotides.
(Item 19)
13. RNA duplex according to item 12, comprising at least one blunt end.
(Item 20)
Item 20. The item 12-19, wherein the RNA duplex comprises an oligonucleotide comprising a nucleotide sequence complementary to a target mRNA, wherein the RNA duplex reduces expression of the target mRNA. RNA duplex.
(Item 21)
21. The RNA duplex of any of items 12-20, wherein the RNA duplex reduces the off-target effect as compared to the same RNA duplex having a natural RNA monomer instead of an acyclic monomer. chain.
(Item 22)
22. The RNA duplex of any of items 12-21, wherein the RNA duplex increases or persists in effectiveness compared to the same RNA duplex having a natural RNA monomer instead of an acyclic monomer. The main chain.
(Item 23)
The RNA duplex of any of items 12-22, wherein the RNA duplex improves stability compared to the same RNA duplex having a natural RNA monomer instead of an acyclic monomer. .
(Item 24)
24. The RNA duplex of any of items 12-23, wherein the RNA duplex reduces immune stimulation compared to the same RNA duplex having a natural RNA monomer instead of an acyclic monomer. .

Claims

Double-stranded RNA containing acyclic Shikimo Nomar.

The acyclic Shikimo Nomar 2'-3'-seco - a nucleotide monomer, RNA duplexes according to claim 1.

In the acyclic monomer, a 2′-hydroxy group is an amino group, an acylated amino group, an alkylated amino group, a dialkylated amino group, a carbamoylated amino group, a piperazino group, an acylated piperazino group, an alkylated piperazino group, 2 ', 3'-seco-nucleotide monomer substituted with a carbamoylated piperazino group, mercapto group, acylated mercapto group, alkylated mercapto group, disulfide group, acylated hydroxy group, alkylated hydroxy group, or carbamoylated hydroxy group The RNA duplex according to claim 1, wherein

The acyclic Shikimo Nomar is motor Nomar D, F, G, H, is selected from the group consisting of I, and J,

Wherein, R, H, alkyl, cholesterol derivatives, fluorophores, polyamines, fatty acids, amino acids, monosaccharides or polypeptide,, RNA duplexes according to claim 1.

The RNA duplex of claim 1, wherein the acyclic monomer is located in a seed region of the antisense strand.

Further comprising 15 to 40 nucleotides and acyclic monomers, RNA duplexes according to claim 1.

Further comprising 1-5 acyclic nucleotide monomer, RNA duplexes according to claim 1.

Further comprising less than four consecutive DNA monomers, RNA duplexes according to claim 1.

Further comprising a RNA monomers greater than 50%, RNA duplexes according to claim 1.

2′-O-alkyl-RNA monomer, 2′-amino-DNA monomer, 2′-fluoro-DNA monomer, LNA monomer, PNA monomer, HNA monomer, ANA monomer, FANA monomer, CeNA monomer, ENA monomer, DNA monomer, and further comprising, RNA duplexes of claim 1 INA monomer or al selection is the nucleotide analogs.

Further comprising at least two LNA nucleotide analogues, RNA duplexes according to claim 1.

Further comprising a phosphorothioate bond or boranophosphate bonds, RNA duplexes according to claim 1.

The RNA duplex is capable of mediating RISC dependent translational repression or degradation of complementary target mRNA in said RNA duplexes, RNA duplexes according to claim 1.

Further comprising 15 to 40 amino number of base pairs, RNA duplexes according to claim 1.

Further comprising 14 to 26 amino number of base pairs, RNA duplexes according to claim 1.

Further comprising at least one projection, RNA duplexes according to claim 1.

Further comprising at least one projection, the length of the protrusion is 1-14 nucleotides, RNA duplexes according to claim 1.

2. The RNA duplex of claim 1 further comprising at least one overhang, wherein the overhang comprises at least one acyclic monomer .

Further comprising at least one 3 'overhang, RNA duplexes according to claim 1.

The RNA duplex of claim 1 , further comprising at least one 3 'overhang, wherein the length of the 3' overhang is 1 to 14 nucleotides.

Further comprising at least one blunt end, RNA duplexes according to claim 1.

Further including a nucleotide sequence complementary to the target specific mRNA, RNA duplexes according to claim 1.

An oligonucleotide comprising an acyclic monomer, wherein the 2'-hydroxy group has an amino group, an acylated amino group, an alkylated amino group, a dialkylated amino group, a carbamoylated amino group, a piperazino group Substituted with acylated piperazino group, alkylated piperazino group, carbamoylated piperazino group, mercapto group, acylated mercapto group, alkylated mercapto group, disulfide group, acylated hydroxy group, alkylated hydroxy group, or carbamoylated hydroxy group Oligonucleotides, which are 2 ′, 3′-seco-nucleotide monomers.

The acyclic nucleotide monomer is selected from the group consisting of the following monomers F, G, H, I, and J;

2. The RNA duplex according to claim 1, wherein R is H, alkyl, cholesterol derivative, fluorophore, polyamine, fatty acid, amino acid, monosaccharide, or polypeptide .

25. A pharmaceutical composition comprising the RNA duplex or oligonucleotide according to any one of claims 1 to 24 and a pharmaceutically acceptable diluent, carrier or adjuvant .

25. A composition for mediating nucleic acid modification of a target nucleic acid in a cell or organism comprising the RNA duplex or oligonucleotide according to any one of claims 1-24.

25. Use of an RNA duplex or oligonucleotide according to any one of claims 1 to 24 in the preparation of a medicament for a therapeutic target .

25. Use of an RNA duplex or oligonucleotide according to any one of claims 1 to 24 in the preparation of a medicament for reducing the expression level of a target mRNA .

25. A composition for the treatment of a disease comprising the RNA duplex or oligonucleotide according to any one of claims 1-24.

A composition for reducing the expression level of a target mRNA, comprising the RNA duplex or oligonucleotide according to any one of claims 1 to 24.